Brazilian Luciano Emilio scored two goals -- one early and one late -- to beat the Revs.
New England, which had won last week's SuperLiga, started three second-year players and five rookies, including 18-year-old Brandon Manzonelli, who made his first professional start, playing the first 75 minutes at forward.
The loss was marked the first time this season that the Revs lost back-to-back games in all competitions. They had gone 25 games without losing two straight matches.
Aug. 12 in Washington, D.C.
D.C. United 3 New England Revolution 1. Goals: Emilio 4, 81, Quaranta 48; Germanese 34.
D.C. United -- Wells, Martinez, McTavish, Peralta, Namoff, Guerrero (Kirk, 86), Simms, Quaranta (Vide, 79), Fred (Zaher, 88), Emilio (Dyachenko, 89), Moreno.
New England -- Warren, Igwe, Flood, Valentino, Thompson, Smith, Joseph, Phelan, Germanese, Cristman, Manzonelli (Tyler, 75).
Red card: New England -- Thompson 71.
Referee: Jorge Gonzalez.